Yahira M. Báez-Santos

Deubiquitinating and Interferon Antagonism Activities of Coronavirus Papain-Like Proteases

ABSTRACT Coronaviruses encode multifunctional proteins that are critical for viral replication and for blocking the innate immune response to viral infection. One such multifunctional domain is the coronavirus papain-like protease (PLP), which processes the viral replicase polyprotein, has deubiquitinating (DUB) activity, and antagonizes the induction of type I interferon (IFN). Here we characterized the DUB and IFN antagonism activities of the PLP domains of human coronavirus NL63 and severe acute respiratory syndrome (SARS) coronavirus to determine if DUB activity mediates interferon antagonism. We found that NL63 PLP2 deconjugated ubiquitin (Ub) and the Ub-line molecule ISG15 from cellular substrates and processed both lysine-48- and lysine-63- linked polyubiquitin chains. This PLP2 DUB activity was dependent on an intact catalytic cysteine residue. We demonstrated that in contrast to PLP2 DUB activity, PLP2-mediated interferon antagonism did not require enzymatic activity. Furthermore, addition of an inhibitor that blocks coronavirus protease/DUB activity did not abrogate interferon antagonism. These results indicated that a component of coronavirus PLP-mediated interferon antagonism was independent of protease and DUB activity. Overall, these results demonstrate the multifunctional nature of the coronavirus PLP domain as a viral protease, DUB, and IFN antagonist and suggest that these independent activities may provide multiple targets for antiviral therapies.

MERS-CoV papain-like protease has deISGylating and deubiquitinating activities

Abstract Coronaviruses encode papain-like proteases (PLpro) that are often multifunctional enzymes with protease activity to process the viral replicase polyprotein and deubiquitinating (DUB)/deISGylating activity, which is hypothesized to modify the innate immune response to infection. Here, we investigate the predicted DUB activity of the PLpro domain of the recently described Middle East Respiratory Syndrome Coronavirus (MERS-CoV). We found that expression of MERS-CoV PLpro reduces the levels of ubiquitinated and ISGylated host cell proteins; consistent with multifunctional PLpro activity. Further, we compared the ability of MERS-CoV PLpro and Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) PLpro to block innate immune signaling of proinflammatory cytokines. We show that expression of SARS-CoV and MERS-CoV PLpros blocks upregulation of cytokines CCL5, IFN-β and CXCL10 in stimulated cells. Overall these results indicate that the PLpro domains of MERS-CoV and SARS-CoV have the potential to modify the innate immune response to viral infection and contribute to viral pathogenesis.

Structural Basis for the Ubiquitin-Linkage Specificity and deISGylating activity of SARS-CoV papain-like protease.

Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes a papain-like protease (PLpro) with both deubiquitinating (DUB) and deISGylating activities that are proposed to counteract the post-translational modification of signaling molecules that activate the innate immune response. Here we examine the structural basis for PLpros ubiquitin chain and interferon stimulated gene 15 (ISG15) specificity. We present the X-ray crystal structure of PLpro in complex with ubiquitin-aldehyde and model the interaction of PLpro with other ubiquitin-chain and ISG15 substrates. We show that PLpro greatly prefers K48- to K63-linked ubiquitin chains, and ISG15-based substrates to those that are mono-ubiquitinated. We propose that PLpros higher affinity for K48-linked ubiquitin chains and ISG15 stems from a bivalent mechanism of binding, where two ubiquitin-like domains prefer to bind in the palm domain of PLpro with the most distal ubiquitin domain interacting with a “ridge” region of the thumb domain. Mutagenesis of residues within this ridge region revealed that these mutants retain viral protease activity and the ability to catalyze hydrolysis of mono-ubiquitin. However, a select number of these mutants have a significantly reduced ability to hydrolyze the substrate ISG15-AMC, or be inhibited by K48-linked diubuiquitin. For these latter residues, we found that PLpro antagonism of the nuclear factor kappa-light-chain-enhancer of activated B-cells (NFκB) signaling pathway is abrogated. This identification of key and unique sites in PLpro required for recognition and processing of diubiquitin and ISG15 versus mono-ubiquitin and protease activity provides new insight into ubiquitin-chain and ISG15 recognition and highlights a role for PLpro DUB and deISGylase activity in antagonism of the innate immune response.

Catalytic Function and Substrate Specificity of the Papain-Like Protease Domain of nsp3 from the Middle East Respiratory Syndrome Coronavirus

ABSTRACT The papain-like protease (PLpro) domain from the deadly Middle East respiratory syndrome coronavirus (MERS-CoV) was overexpressed and purified. MERS-CoV PLpro constructs with and without the putative ubiquitin-like (UBL) domain at the N terminus were found to possess protease, deubiquitinating, deISGylating, and interferon antagonism activities in transfected HEK293T cells. The quaternary structure and substrate preferences of MERS-CoV PLpro were determined and compared to those of severe acute respiratory syndrome coronavirus (SARS-CoV) PLpro, revealing prominent differences between these closely related enzymes. Steady-state kinetic analyses of purified MERS-CoV and SARS-CoV PLpros uncovered significant differences in their rates of hydrolysis of 5-aminomethyl coumarin (AMC) from C-terminally labeled peptide, ubiquitin, and ISG15 substrates, as well as in their rates of isopeptide bond cleavage of K48- and K63-linked polyubiquitin chains. MERS-CoV PLpro was found to have 8-fold and 3,500-fold higher catalytic efficiencies for hydrolysis of ISG15-AMC than for hydrolysis of the Ub-AMC and Z-RLRGG-AMC substrates, respectively. A similar trend was observed for SARS-CoV PLpro, although it was much more efficient than MERS-CoV PLpro toward ISG15-AMC and peptide-AMC substrates. MERS-CoV PLpro was found to process K48- and K63-linked polyubiquitin chains at similar rates and with similar debranching patterns, producing monoubiquitin species. However, SARS-CoV PLpro much preferred K48-linked polyubiquitin chains to K63-linked chains, and it rapidly produced di-ubiquitin molecules from K48-linked chains. Finally, potent inhibitors of SARS-CoV PLpro were found to have no effect on MERS-CoV PLpro. A homology model of the MERS-CoV PLpro structure was generated and compared to the X-ray structure of SARS-CoV PLpro to provide plausible explanations for differences in substrate and inhibitor recognition. IMPORTANCE Unlocking the secrets of how coronavirus (CoV) papain-like proteases (PLpros) perform their multifunctional roles during viral replication entails a complete mechanistic understanding of their substrate recognition and enzymatic activities. We show that the PLpro domains from the MERS and SARS coronaviruses can recognize and process the same substrates, but with different catalytic efficiencies. The differences in substrate recognition between these closely related PLpros suggest that neither enzyme can be used as a generalized model to explain the kinetic behavior of all CoV PLpros. As a consequence, decoding the mechanisms of PLpro-mediated antagonism of the host innate immune response and the development of anti-CoV PLpro enzyme inhibitors will be a challenging undertaking. The results from this study provide valuable information for understanding how MERS-CoV PLpro-mediated antagonism of the host innate immune response is orchestrated, as well as insight into the design of inhibitors against MERS-CoV PLpro.

The SARS-coronavirus papain-like protease: Structure, function and inhibition by designed antiviral compounds

Abstract Over 10 years have passed since the deadly human coronavirus that causes severe acute respiratory syndrome (SARS-CoV) emerged from the Guangdong Province of China. Despite the fact that the SARS-CoV pandemic infected over 8500 individuals, claimed over 800 lives and cost billions of dollars in economic loss worldwide, there still are no clinically approved antiviral drugs, vaccines or monoclonal antibody therapies to treat SARS-CoV infections. The recent emergence of the deadly human coronavirus that causes Middle East respiratory syndrome (MERS-CoV) is a sobering reminder that new and deadly coronaviruses can emerge at any time with the potential to become pandemics. Therefore, the continued development of therapeutic and prophylactic countermeasures to potentially deadly coronaviruses is warranted. The coronaviral proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro), are attractive antiviral drug targets because they are essential for coronaviral replication. Although the primary function of PLpro and 3CLpro are to process the viral polyprotein in a coordinated manner, PLpro has the additional function of stripping ubiquitin and ISG15 from host-cell proteins to aid coronaviruses in their evasion of the host innate immune responses. Therefore, targeting PLpro with antiviral drugs may have an advantage in not only inhibiting viral replication but also inhibiting the dysregulation of signaling cascades in infected cells that may lead to cell death in surrounding, uninfected cells. This review provides an up-to-date discussion on the SARS-CoV papain-like protease including a brief overview of the SARS-CoV genome and replication followed by a more in-depth discussion on the structure and catalytic mechanism of SARS-CoV PLpro, the multiple cellular functions of SARS-CoV PLpro, the inhibition of SARS-CoV PLpro by small molecule inhibitors, and the prospect of inhibiting papain-like protease from other coronaviruses. This paper forms part of a series of invited articles in Antiviral Research on “From SARS to MERS: 10years of research on highly pathogenic human coronaviruses.”

X-ray Structural and Biological Evaluation of a Series of Potent and Highly Selective Inhibitors of Human Coronavirus Papain-like Proteases.

Structure-guided design was used to generate a series of noncovalent inhibitors with nanomolar potency against the papain-like protease (PLpro) from the SARS coronavirus (CoV). A number of inhibitors exhibit antiviral activity against SARS-CoV infected Vero E6 cells and broadened specificity toward the homologous PLP2 enzyme from the human coronavirus NL63. Selectivity and cytotoxicity studies established a more than 100-fold preference for the coronaviral enzyme over homologous human deubiquitinating enzymes (DUBs), and no significant cytotoxicity in Vero E6 and HEK293 cell lines is observed. X-ray structural analyses of inhibitor-bound crystal structures revealed subtle differences between binding modes of the initial benzodioxolane lead (15g) and the most potent analogues 3k and 3j, featuring a monofluoro substitution at para and meta positions of the benzyl ring, respectively. Finally, the less lipophilic bis(amide) 3e and methoxypyridine 5c exhibit significantly improved metabolic stability and are viable candidates for advancing to in vivo studies.

Discovery of N-(benzo[1,2,3]triazol-1-yl)-N-(benzyl)acetamido)phenyl) carboxamides as severe acute respiratory syndrome coronavirus (SARS-CoV) 3CLpro inhibitors: Identification of ML300 and noncovalent nanomolar inhibitors with an induced-fit binding

Abstract Herein we report the discovery and SAR of a novel series of SARS-CoV 3CLpro inhibitors identified through the NIH Molecular Libraries Probe Production Centers Network (MLPCN). In addition to ML188, ML300 represents the second probe declared for 3CLpro from this collaborative effort. The X-ray structure of SARS-CoV 3CLpro bound with a ML300 analog highlights a unique induced-fit reorganization of the S2–S4 binding pockets leading to the first sub-micromolar noncovalent 3CLpro inhibitors retaining a single amide bond.

X-ray Structure and Enzymatic Activity Profile of a Core Papain-like Protease of MERS Coronavirus with utility for structure-based drug design.

Ubiquitin-like domain 2 (Ubl2) is immediately adjacent to the N-terminus of the papain-like protease (PLpro) domain in coronavirus polyproteins, and it may play a critical role in protease regulation and stability as well as in viral infection. However, our recent cellular studies reveal that removing the Ubl2 domain from MERS PLpro has no effect on its ability to process the viral polyprotein or act as an interferon antagonist, which involves deubiquitinating and deISGylating cellular proteins. Here, we test the hypothesis that the Ubl2 domain is not required for the catalytic function of MERS PLpro in vitro. The X-ray structure of MERS PLpro-∆Ubl2 was determined to 1.9 Å and compared to PLpro containing the N-terminal Ubl2 domain. While the structures were nearly identical, the PLpro-∆Ubl2 enzyme revealed the intact structure of the substrate-binding loop. Moreover, PLpro-∆Ubl2 catalysis against different substrates and a purported inhibitor revealed no differences in catalytic efficiency, substrate specificity, and inhibition. Further, no changes in thermal stability were observed between enzymes. We conclude that the catalytic core of MERS PLpro, i.e. without the Ubl2 domain, is sufficient for catalysis and stability in vitro with utility to evaluate potential inhibitors as a platform for structure-based drug design.

Formulation and characterization of a liquid crystalline hexagonal mesophase region of phosphatidylcholine, sorbitan monooleate, and tocopherol acetate for sustained delivery of leuprolide acetate

Although liquid crystal (LC) systems have been studied before, their utility in drug delivery applications has not been explored in depth. This study examined the development of a 1-month sustained release formulation of leuprolide acetate using an in situ-forming LC matrix. The phase progression upon water absorption was tested through construction of ternary phase diagrams of phosphatidylcholine, sorbitan monooleate, and tocopherol acetate (TA) at increasing water content. Small angle X-ray scattering revealed the presence of lamellar and hexagonal mesophases. The physicochemical characteristics and in vitro drug release were evaluated as a function of the ternary component ratio and its resultant phase behavior. Formulations with increased water uptake capacity displayed greater drug release and enhanced erodability. Removal of TA resulted in increased water uptake capacity and drug release, where 8% (w/w) TA was determined as the critical concentration threshold for divergence of release profiles. In conclusion, characterization of the resultant HII mesophase region provided information of the impact the individual components have on the physicochemical properties and potential drug release mechanisms. This high mitigating impact of TA on drug release indicates the use of TA as a tailoring agent, broadening the therapeutic applications of this LC system.

The in vivo transformation and pharmacokinetic properties of a liquid crystalline drug delivery system

A liquid crystalline (LC) system, composed of phosphatidylcholine, sorbitan monoleate, and tocopherol acetate, was investigated to understand the in vivo transformation after subcutaneous injection, coupled with the physicochemical and pharmacokinetic properties of the formulation. The rat model was utilized to monitor a pseudo-time course transformation from a precursor LC formulation to the LC matrix, coupled with the blood concentration profiles of the formulations containing leuprolide acetate. Three formulations that result in the HII phase, demonstrating dissimilar in vitro release profiles, were used. The formulation showing the highest AUC, Cmax and Tmax, also displayed the greatest release rate in vitro, the lowest viscosity (LC matrix), and an earlier transformation (LC precursor to matrix) in vivo. A potential link between viscosity, phase transformation, and drug release properties of a liquid crystalline system is described.